The tissue-specific transcription factor, Pit-l, has previously been cloned and shown to regulate two hormones secreted by the pituitary, growth hormone (GH) and prolactin (PRL). Further, Pit-1 transcripts are found in both cell types and appear one day prior to the onset of GH and PRL gene expression. Yet, both of these cell types are able to restrict their gene expression to only one unique hormone. The molecular mechanisms for this additional specificity have not been identified. While the correlation between Pit-1 and GH/PRL gene expression is striking, Pit-1 transcripts and protein are also found in another pituitary cell type, thyrotrophs, known to secrete thyrotropin (TSH). The overall aim of this proposal is to investigate molecular mechanisms responsible for establishing a subset of cell types in the anterior pituitary by defining the combinatorial effectors that interact with Pit-l in normal pituitary development. Several parallel and complementing strategies have been initiated in order to start defining this combinatorial code. The study proposes to identify cellular proteins that interact with Pit-l by a screening method using radiolabeled Pit-1 or its sub-domains to screen an expression library. Two independent yeast screening strategies will attempt to isolate proteins that directly or indirectly interact with Pit-l. Proteins that interact with Pit-l will be characterized as to their patterns of pituitary expression, and the functional consequences of this interaction by in vivo and in vitro analyses.